Spiders are ecologically important predators with complex venom and extraordinarily tough silk that enables capture of large prey. Here we present the assembled genome of the social velvet spider and a draft assembly of the tarantula genome that represent two major taxonomic groups of spiders. The spider genomes are large with short exons and long introns, reminiscent of mammalian genomes. Phylogenetic analyses place spiders and ticks as sister groups supporting polyphyly of the Acari. Complex sets of venom and silk genes/proteins are identified. We find that venom genes evolved by sequential duplication, and that the toxic effect of venom is most likely activated by proteases present in the venom. The set of silk genes reveals a highly dynamic gene evolution, new types of silk genes and proteins, and a novel use of aciniform silk. These insights create new opportunities for pharmacological applications of venom and biomaterial applications of silk.

Matrix metalloproteinases (MMPs) and interleukin 1 (IL-1) are implicated in inflammation and tissue destruction, where IL-1 is a potent stimulator of connective tissue cells to produce the extracellular matrix-degrading MMPs. Here, we report that IL-1␤, but not IL-1␣, is degraded by MMP-1 (interstitial collagenase), MMP-2 (gelatinase A), MMP-3 (stromelysin 1), and MMP-9 (gelatinase B). This degradation was effectively blocked by tissue inhibitor of metalloproteinases (TIMP)-1. When IL-1␤ was treated with MMPs it lost the ability to enhance the synthesis of prostaglandin E 2 and pro-MMP-3 in human fibroblasts. The primary cleavage site of IL-1␤ by MMP-2 was identified at the Glu 25 -Leu 26 bond. These results suggest that IL-1␤ stimulates connective tissue cells to produce MMPs, but activated MMPs in turn negatively regulate the activity of IL-1␤. Matrix metalloproteinases (MMPs),1 also called matrixins, degrade extracellular matrix macromolecules and play important roles in many biological processes such as morphogenesis, ovulation, embryo implantation, cell migration, tissue involution, angiogenesis, and wound healing (1-3). In excess, they participate in the destruction of the tissue associated with many connective tissue diseases such as arthritis, periodentitis, nephritis, and tissue ulcerations and with tumor cell invasion and metastasis (1-3). The importance of matrixins in both physiological and pathological catabolism of extracellular matrix macromolecules has been emphasized because little MMP activities can be detected in normal steady state tissue, but the synthesis of many MMPs is transcriptionally regulated by inflammatory cytokines, hormones, growth factors, and cellular transformation (1-3). For example, high levels of MMP-1 (interstitial collagenase, EC 3.4.24.7), MMP-3 (stromelysin 1, EC 3.4.24.17), and MMP-9 (gelatinase B, EC 3.4.24.35) are found in synovial tissues and fluids from patients with rheumatoid arthritis (4 -6). It is generally accepted that an elevated level of interleukin 1 (IL-1) is one of the key mediators that greatly enhances the biosynthesis and secretion of precursors of these MMPs (pro-MMPs) and prostaglandin E 2 from mesenchymal cells at inflammatory sites (1).IL-1 is secreted from activated macrophages and a variety of other cell types and elicits many other biological responses such as thymocyte proliferation, fever production, wound healing, and tissue resorption (see Ref. 7 for review). The promotion of wound healing and tissue degradation is considered to be in part due to the production of MMPs by cells stimulated with IL-1. The suppression of IL-1 activity is, therefore, thought to be an effective step to control inflammatory responses. In this regard, a large number of studies have focused on the regulation of IL-1 synthesis, processing of the IL-1 precursor, and the receptor antagonist (7). However, little is known about the catabolism of the mature form of IL-1.In this communication, we report that MMP-1, MMP-2 (gelatinase A, EC 3.4.24.24), MMP-3, and MMP-9 secre...

Proteolytic enzymes produced by Porphyromonas gingivalis are important virulence factors of this periodontopathogen. Two of these enzymes, referred to as arginine-specific cysteine proteinases (gingipains R), are the product of two related genes. Here, we describe the purification of an enzyme translated from the rgpB/rgp-2 gene (gingipain R2, RGP-2) and secreted as a single chain protein of 422 residues. The enzyme occurs in several isoforms differing in pI, molecular mass, mobility in gelatin zymography gels, and affinity to arginineSepharose. In comparison to the 95-kDa gingipain R1, a complex of catalytic and hemagglutinin/adhesin domains, RGP-2 showed five times lower proteolytic activity, although its activity on various P 1 -arginine p-nitroanilide substrates was generally higher. Gingipains R amidolytic activity, but not general proteolytic activity, was stimulated by glycyl-glycine. However, in cases of limited proteolysis, such as the inactivation of ␣-1-antichymotrypsin, glycyl-glycine potentiated inhibitor cleavage. In contrast, ␣-1-proteinase inhibitor was not inactivated by gingipains R and only underwent proteolytic degradation during boiling in reducing SDS-polyacrylamide gel electrophoresis treatment buffer. Similarly, native type I collagen was completely resistant to cleavage by gingipains but readily degraded after denaturation. Together, these data explain much of the controversy regarding gingipains structure and substrate specificity and indicate that these enzymes function as P. gingivalis virulence factors by proteolysis of selected target proteins rather than random degradation of host connective tissue components.

Aggrecanases have been characterized as proteinases that cleave the Glu 373 -Ala 374 bond of the aggrecan core protein, and they are multidomain metalloproteinases belonging to the ADAMTS (adamalysin with thrombospondin type 1 motifs) family. The first aggrecanases discovered were ADAMTS-4 (aggrecanase 1) and ADAMTS-5 (aggrecanase 2). They contain a zinc catalytic domain followed by non-catalytic ancillary domains, including a disintegrin domain, a thrombospondin domain, a cysteine-rich domain, and a spacer domain. In the case of ADAMTS-5, a second thrombospondin domain follows the spacer domain. We previously reported that the non-catalytic domains of ADAMTS-4 influence both its extracellular matrix interaction and proteolytic abilities. Here we report the effects of these domains of ADAMTS-5 on the extracellular matrix interaction and proteolytic activities and compare them with those of ADAMTS-4. Although the spacer domain was critical for ADAMTS-4 localization in the matrix, the cysteine-rich domain influenced ADAMTS-5 localization. Similar to previous reports of other ADAMTS family members, very little proteolytic activity was detected with the ADAMTS-5 catalytic domain alone. The sequential inclusion of each carboxyl-terminal domain enhanced its activity against aggrecan, carboxymethylated transferrin, fibromodulin, decorin, biglycan, and fibronectin. Both ADAMTS-4 and -5 had a broad optimal activity at pH 7.0 -9.5. Aggrecanolytic activities were sensitive to the NaCl concentration, but activities on non-aggrecan substrates, e.g. carboxymethylated transferrin, were not affected. Although ADAMTS-4 and ADAMTS-5 had similar general proteolytic activities, the aggrecanase activity of ADAMTS-5 was at least 1,000-fold greater than that of ADAMTS-4 under physiological conditions. Our studies suggest that ADAMTS-5 is a major aggrecanase in cartilage metabolism and pathology.Destruction of articular cartilage is a feature of various arthritides, including rheumatoid and osteoarthritis, that results in joint impairment and disability. It is caused primarily by an elevation in proteolytic enzymes that degrade macromolecules of the cartilage extracellular matrix. Aggrecan degradation is initially observed followed by essentially irreversible collagen degradation. The proteinases that are responsible for aggrecan degradation in cartilage are matrix metalloproteinases (MMPs) 4 and "aggrecanases," members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin type 1 motifs) family (1, 2). Aggrecanase activity was first defined as the ability to cleave the Glu 373 -Ala 374 bond in the interglobular domain (IGD) of the aggrecan core protein (3, 4). The first two proteinases shown to be capable of cleaving aggrecan at this site were ADAMTS-4 (aggrecanase 1) (5), and ADAMTS-5 5 (aggrecanase 2) (6). More recently, ADAMTS-1, -8, -9, -15, -16, and -18 were shown to cleave the Glu 373 -Ala 374 bond in the IGD at a high enzyme-substrate ratio (2, 7). Although this bond is also cleaved by MMP-8 (8) and MMP-14 (9) a...